Composition comprising at least one detergent component and at least one enzyme component for removing biofilms

ABSTRACT

The invention relates to a composition for removing biofilms, said composition comprising at least one enzyme component and at least one detergent component, the at least one enzyme component containing β-1,6-N-acetylglucosaminidase, and the detergent component containing at least one wetting agent, at least one chelating agent and at least one dispersing agent.

The present invention relates to a composition for removing biofilms,said composition comprising at least one enzyme component and at leastone detergent component.

Such a composition is known from document EP2243821 which discloses acomposition for removing biofilms present on a substrate, thiscomposition comprising (1) a detergent component simultaneouslycontaining a wetting agent, a sequestering agent and a dispersing agentand (2) comprising an enzyme component simultaneously containing atleast one protease, at least one laccase and at least onepolysaccharidase. Such a composition is described as being able to beused to remove biofilms in installations, for cleaning-in-place orcleaning-by-soaking floors or surfaces, for cleaning-in-place orcleaning-by-soaking surgical tools and for cleaning in the foodindustry.

A biofilm is a slime layer which grows on all surfaces, following theadhesion of microorganisms on these surfaces and the secretion therefromof polymers which coat them and facilitate their adhesion. The biofilmsthus form a layer of protection around the microorganisms and representa recurrent source of contamination of the surrounding environment whichposes problems, for example in the food industry and in hospitalenvironments.

More specifically, the accumulation of polymers secreted by the bacteriacreates a matrix essentially formed of polysaccharides, DNA, proteins aswell as lipids, which protects these microorganisms from externalaggressions and is very resistant to conventional cleaning anddisinfecting procedures. The microorganisms therefore develop easilywithin this protective matrix and contaminate the surroundingenvironment by forming a particularly critical reservoir which isdifficult to eliminate.

It is known that the problem of the presence of biofilms is twofold.Firstly, as indicated above, they represent a permanent source ofcontamination which is very difficult to eliminate by conventionalmeans, even by the most aggressive means. Indeed, common disinfectantsare often ineffective as it has been observed that they do not reach themicroorganisms which are protected by the biofilm matrix formed ofpolysaccharides, DNA, proteins and lipids.

Secondly, a biofilm is generally mixed, in that it is initiallydeveloped by certain bacterial strains, but it may accommodate others,these strains living and developing in colonies. However, these coloniespromote communication between bacteria and, among other things, theexchange and spreading of resistance genes carried by certain bacteria.The biofilms formed by these gene exchanges are thus more difficult toeliminate and increasingly powerful means of disinfection or treatmentmust be resorted to, which, however, frequently face major problems ofresistance and/or tolerance.

The protective matrix of the bacteria forming the biofilms is soresistant that it constitutes a real barrier protecting the bacteriafrom dehydration phenomena, antibiotic and biocidal action (and moregenerally, microbicidal molecules), phagocytosis and acids.

In hospital and veterinary environments, but also in agri-foodenvironments, the situation is all the more critical as numerousmicroorganisms responsible for the formation of biofilms are detected innumerous places. In hospital and veterinary offices, biofilms aredetected around individuals (patients and animals) as well as in thesurrounding area (operating room, surgical tools, equipment formaintaining said tools, endoscopes, urinary tubes, catheters, medicalequipment, dialysis or assisted breathing machines for individuals,etc.) and on surfaces (floors, walls, operating tables, etc.). In theagri-food field, and particularly in factories for manufacturing foodproducts, biofilms can be found around machines, tables, packaging andon operators even if they take all precautions possible in order toavoid contaminating the surfaces and work tools.

From all of this, it appears that biofilms constitute a real problem,particularly in the fields of healthcare (hospitals, dental practices,etc.), veterinary care and agri-food. This problem is all the morecritical as biofilms may involve bacteria responsible for infectionswhich could be fatal in individuals, if these bacteria are present inhospitals, veterinary practices or in food products. It is thereforenecessary to take all possible precautions in order to avoid theformation and development of biofilms.

These days, in order to fight against biofilms, enzyme formulationscomprising a detergent base are being implemented, as proposed indocument EP2243821. The majority of the current formulations are basedmore particularly on a combination of several enzymes, theseformulations still typically comprising at least one protease. However,even the formulations (compositions) combining a detergent base andseveral enzymes have certain limits in terms of removing biofilms from asubstrate (from a surface).

Unfortunately, it therefore currently appears that, even if acomposition such as that disclosed in document EP2243821 proves to beeffective in terms of removing biofilms, the compositions for removingbiofilms too often depend on the use of an enzyme cocktail. Morespecifically, with respect to document EP2243821, the composition forremoving biofilms disclosed depends on the use of at least three enzymes(protease and laccase and polysaccaridase) formulated in a detergentbase, itself using three different detergent agents (sequestering andwetting and dispersing). It is therefore important, to formulate acomposition for removing biofilms according to document EP2243821, touse at least six different components, which offers little economicadvantage as handling different components to be formulated togetherrequires time and these six components must be obtained at the sametime.

However, it is known that, in a formulation comprising several enzymesincluding a protease, the latter is inevitably going to graduallydeteriorate the other enzymes present over time and forcibly reducetheir enzymatic activity over time, this therefore having an impact onthe stability and effectiveness of the formulations comprising aprotease. Furthermore, it appears that the current compositions havecertain limits, particularly with respect to removing “old” biofilms,that is biofilms that have been in place for months or years.

The object of the invention is to overcome the disadvantages of theprior art by obtaining a composition for removing biofilms which iseasier to formulate (to use), whose stability and effectiveness arepreserved over time and which allows effective action to be takenagainst all types of biofilm, including “old” biofilms.

To at least partially solve the problems of the prior art, a compositionaccording to the invention is provided for removing biofilms asindicated above, characterised in that said at least one enzymecomponent comprises β-1,6-N-acetylglucosaminidase and in that saiddetergent component contains at least one wetting agent, at least onesequestering agent and at least one dispersing agent.

According to the present invention, the enzyme component may compriseβ-1,6-N-acetylglucosaminidase as the sole enzyme or the same enzymecombined with others.

It should be noted that, according to EC nomenclature (Enzyme Commissionnumbers), β-1,6-N-acetylglucosaminidase forms part of the EC3.2.1-class, in contrast to β-N-acetylglucosaminidase (orhexosaminidase) and β-N-acetylhexosaminidase which form part of the EC3.2.1.52 class or endo-β-N-acetylglucosaminidase forming part of the EC3.2.1.96 class.

β-1,6-N-acetylglucosaminidase is active on β-1,6-type bonds whileβ-N-acetylglucosaminidase (or hexosaminidase) andβ-N-acetylhexosaminidase are active on β-1,4-type bonds. In particular,β-1,6-N-acetylglucosaminidase splits the polymerpoly-β-1,6-N-acetyl-D-glucosamide (PNAG) which is an exopolysaccharide.

In an entirely surprising and unexpected manner, it has been shown, inthe scope of the present invention, that such a composition according tothe invention comprising at least β-1.6-N-acetylglucosaminidase in atleast one detergent component which comprises at least one wetting agentand at least one sequestering agent and at least one dispersing agent isat least as effective as a composition according to document EP2243821despite, as indicated above, this composition of the prior art dependingon the joint use of three enzymes in the same detergent component. Thismeans that, according to the present invention, an entirely effectivecomposition in terms of removing biofilms from a surface may be preparedby only using β-1,6-N-acetylglucosaminidase in place of an enzymecocktail depending on the simultaneous presence of three differentenzymes. The formulation of a composition according to the invention isconsequently easier as it requires the use of fewer differentcomponents, which is more economically viable.

In an even more surprising and unexpected manner, it has beendetermined, within the scope of the present invention, that the threedetergent agents of the detergent component have a synergic effect byoptimising the effectiveness of β-1,6-N-acetylglucosaminidase in termsof removing the biofilm but also in terms of enzyme activity of thelatter over time. On the one hand, it has indeed been determined thatthis particular combination of three detergent agents (at least onewetting agent+at least one sequestering agent+at least one dispersingagent) ensures significantly higher enzyme activity ofβ-1,6-N-acetylglucosaminidase over time with respect to the enzymeactivities identified for this same enzyme when the detergent componentonly comprises, for example, two of these three detergent agents. On theother hand, it has been highlighted that this particular combination ofthree detergent agents increases the effectiveness of the compositionaccording to the invention: optimum removal of the biofilms is observedwhen β-1,6-N-acetylglucosaminidase is formulated in a detergentcomponent simultaneously comprising at least one wetting agent, at leastone sequestering agent and at least one dispersing agent as opposed toin a detergent component only comprising, for example, two of thesethree detergent agents.

Furthermore, with a composition according to the invention and unlike acomposition according to document EP2243821, the presence of a proteaseis not necessarily required in order to obtain adequate removal ofbiofilms from the surfaces, this allowing the problem described above tobe eliminated, and linked to the presence of this enzyme in aformulation where several enzymes are present.

In addition, unlike the compositions for removing biofilms known in theprior art, it has been demonstrated that a composition according to theinvention, comprising at least β-1,6-N-acetylglucosaminidase in at leastone detergent component containing at least one wetting agent and atleast one sequestering agent and at least one dispersing agent, allowseffective action to be taken, even on “old” biofilms which areparticularly difficult to remove.

In the scope of the present invention, it has been shown that, followingpossible detection of the presence of biofilms, for example, using adetection kit as described in document EP2537601, treatment with acomposition according to the invention allowsβ-1,6-N-acetylglucosaminidase to effectively and versatilely deterioratethe organic polymers of different natures forming the matrix of biofilmsformed by a multitude of different microorganisms.

Under the action of β-1,6-N-acetylglucosaminidase and together with theaction of the detergent component, the biofilm matrix is weakened andinflated, which allows it to be removed from the treated surface.Furthermore, surprisingly, it has also been shown that the compositionaccording to the invention is not specific to a particularmicroorganism, and thus to a particular type of biofilm, but that it isadapted to numerous bacterial strains.

The detergent action of the composition according to the inventionfurther allows the effectiveness of the composition according to theinvention to be ensured. To that end, a detergent base (detergentcomponent) according to the invention is provided which is compatibleand which can act in a synergic manner with the enzyme activity of theenzyme component. Furthermore, according to the invention, a detergentbase which allows the rapidity and effectiveness of the biofilm removalto be significantly improved is provided. It is for these reasons thatthe present invention combines a wetting agent and a dispersing agentand a sequestering agent. The joint actions of these three agents of thedetergent component of the composition according to the invention allowthe surface portion of the biofilm to be removed, to wet and inflate theorganic structures of the biofilm in this way, consequently furtheringthe accessibility of the enzyme component which, in turn, weakens anddeteriorates the biofilm matrix.

According to the present invention, the detergent component comprises atleast one wetting agent and at least one dispersing agent and at leastone sequestering agent. As indicated above, this detergent componentacts, first of all, by removing a surface portion of the biofilm, thisby wetting and/or inflating the organic structures of the biofilm. Inthis way, the detergent component furthers the accessibility of theenzyme component by breaking down the biofilm matrix. The enzymecomponent then acts in synergy with the detergent component and weakensand breaks down the matrix of the biofilm in turn. This action combinedwith the enzyme component and the detergent component, perfectlycompatible with correct action of β-1,6-N-acetylglucosaminidase,furthers the accessibility of the composition to the deeper layers ofthe biofilms and allows a quick and optimal detachment of any kind ofbiofilm while preserving the treated substrate (the treated surface).Thus, the detergent component allows the enzymes to act quickly on allbiofilm structures.

The dispersing agent of the detergent component allows the separation ofthe particles of a suspension to be improved in order to preventagglutination, aggregation and/or decantation. This dispersing agent maybe a polymer which is soluble or partially soluble in water such as, forexample, polyethylene glycol, cellulose derivatives or a polymercomprising at least one acrylic acid or acrylic ester or polyphosphateunit. Preferentially, the dispersing agent is a polymer comprising atleast one acrylic acid or acrylic ester unit of general formula—(CH₂—CH—COOR)—, wherein R represents an alkyl or substituted alkyl,aryl or substituted aryl, or hydrogen group. For example, the dispersingagent is a polymer having an average molecular weight Mw rangingapproximately between 500 and 10,000.

More preferentially, the dispersing agent is an acrylic acid polymer.For example, the dispersing agent may be an acrylic acid homopolymerhaving an average molecular weight ranging approximately between 2,000and 6,000.

The presence of a dispersing agent in the composition according to theinvention thus allows any aggregation of bacterial particles duringcleaning of surfaces to be avoided, which ensures an optimal removal ofthe particles of biofilms detached from a substrate under the action ofthe enzymes. In fact, rather than joining together, these particlesremain separated in a suspension, not re-depositing or re-adhering tothe cleaned surface.

The wetting agent of the detergent component is an amphiphilic chemicalsubstance, or a composition comprising said amphiphilic chemicalsubstance, which modifies the surface tension between two surfaces. Thewetting agent has the advantage of promoting the spreading of a liquidon a solid but also of increasing the contact between two surfaces. Moreparticularly, the wetting agent has the advantage of furthering contactbetween the detergent component and a surface and, consequently, betweenthe enzymes and their substrate. For example, on stainless steelsurfaces frequently found in the agri-food industries but also inhospital or veterinary environments, the wetting agent allowshomogeneous spreading of the composition to be carried out, and thus itsperfect distribution on the surfaces to be decontaminated, for exampleon production tools, workplans, floors, or operating tables and medicaltools.

The wetting agent may be anionic, cationic, non-ionic or zwitterionic.Preferentially, the wetting agent may be an anionic or non-ionic wettingagent, that is the hydrophilic part is negatively charged or bears nonet charge, or may be a composition comprising an anionic wetting agent.More particularly, the wetting agent may be a sucrose ester or acomposition comprising a sodium alkyl sulphate and an alcohol.

The wetting agent may be foaming or non-foaming. Preferably, in thedetergent component according to the invention, said wetting agent isnot hot-foaming and is preferably chosen from the group of C₆ to C₁₂sodium alkyl sulphates, C₆ to C₁₀ etheric alcohol sulphates and C₆ toC₁₂ alkyl aryl sulphonates.

The sequestering agent is a chemical substance having the capacity toform complexes with mineral ions which it arranges in a form preventingtheir precipitation by standard reactions. For example, the sequesteringagent may be ethylene-diamine-tetraacetic acid, glucono-delta-lactone,sodium gluconate, potassium gluconate, calcium gluconate, citric acid,phosphoric acid, tartaric acid, sodium acetate, sorbitol, a compoundcomprising a phosphorous atom. Preferentially, the sequestering agentmay be a phosphorous oxide such as phosphonate, phosphinate or phosphateor mixtures thereof, or a salt thereof, an amine or an amine oxidehaving at least, in its structure, a functional phosphine group,phosphine oxide, phophinite, phosphonite, phosphite, phosphonate,phosphinate or phosphate, alone or in combination, or a salt thereof.

More preferentially, the sequestering agent may be a phosphonate or asalt there of, an amine or an amine oxide comprising at least, in itsstructure, a functional phosphine group, phosphine oxide, phosphinite,phosphonite, phosphite, phosphonate, phosphinate or phosphate, alone orin combination, or a salt thereof. As a non-limiting example, thephosphonate may be of general formula R¹(R²O)(R³O)P═O, wherein R¹, R²and R³ independently represent a hydrogen, alkyl, substituted alkyl,substituted alkyl-amino or not, substituted amino-alkyl or not, aryl orsubstituted aryl group. As a non-limiting example, the amine or amineoxide may comprise one, two or three substitute(s) of general formulaCR⁴R⁵W, wherein R⁴ and R⁵ represent, independently from one another, ahydrogen, alkyl, substituted alkyl, substituted or non-substitutedalkyl-amino, substituted or non-substituted amino-alkyl, aryl or arylsubstituted group, and W represents a phosphonate, phosphinate orphosphate group.

The sequestering agent may be in the form of a sodium, calcium, lithium,magnesium or potassium salt; preferentially, the sequestering agent maybe in the form of a sodium, calcium or potassium salt.

Preferably, the sequestering agent is an agent which may be used safelyin the food industry, in that the sequestering agent is no risk tohealth, alone or combined with other components.

Advantageously, according to the invention, said at least one enzymecomponent comprises at least one additional enzyme chosen from the groupconstituted of α-polysaccharidases (lactase, amylase, α-glucosidase,etc.), β-polysaccharidases (cellulase, hemi-cellulase, β-glucanase,arabanase, pectinase, chitinase, xylanase, dextranase, lysozyme,pullulanase, β-glucisidase, mannanase, etc.), oxidoreductases (laccase,etc.), lyases (lyase pectate, etc.), transferases, proteases andpeptidases (metalloprotease, serine-proteases, exopeptidase,endoprotease, cysteine protease, etc.), lipases and esterases(lysophospholipase phospholipase, etc.).

Preferably, the composition for the removal of biofilms according to theinvention has a pH ranging between 5 and 11.

In a preferred manner, according to the invention, said at least onedetergent component comprises a proportion of sequestering agent between1 and 10 wt %, a proportion of dispersing agent between 1 and 10 wt %and a proportion of wetting agent between 1 and 30 wt % with respect tothe total weight of the detergent component. Preferentially, theproportion of wetting agent between 5 and 20 wt % with respect to thetotal weight of the detergent component. In a preferred manner, theproportion of wetting agent is 15 wt % with respect to the total weightof the detergent component.

Advantageously, according to the invention, said at least one enzymecomponent and said at least one detergent component are in solution in asolvent so as to form a vaporisable solution.

Preferentially, according to the invention, said at least one enzymecomponent and said at least one detergent component are present in solidform, for example, in the form of a lyophilisate, a powder, granules orin any other form soluble in a solvent.

Preferentially, said at least one enzyme component is a solution whosepH may range between approximately 8 and 10.

Preferentially, said at least one enzyme component is an aqueoussolution whose pH may range between approximately 5 and 11; morepreferentially the pH may be approximately between 7 and 10, and this topreserve the integrity of the enzymes as much as possible.

Alternatively, said at least one enzyme may be in a solid form such as,for example, the form of a lypophilisate, powder, granules or in anyother form allowing solubilisation of said component in a solvent, thenit will be later dissolved in said solvent. The solvent may be water oran aqueous, acidic, basic, alcoholic, buffer or neutral solution. Saidat least one soluble enzyme component could then in this case be laterdiluted in an aqueous solution optionally containing one or severalcompounds such as, for example, detergents to form the cleaningsolution.

As for said at least one enzyme component, said at least one detergentcomponent may be in a solid form to dissolve in a solvent and/or in anaqueous phase, or in liquid form.

While it is in a solid form, it may either be dissolved directly intothe solution formed by the enzyme component optionally already dilutedin the aqueous phase, or be dissolved in a solvent, prior to itsdilution in the solution formed by the enzyme component and the aqueousphase, or directly in the aqueous phase, before the dilution of theenzyme component.

When said at least one detergent component is in a liquid form, 100% ofthe detergent components are optionally obtained generally using waterand, prior to the application to the biofilm, it will be diluted in anaqueous phase, optionally already containing the enzyme compound.

Other embodiments of a composition according to the invention areindicated in the appended claims.

The object of the invention also includes a method for removing biofilmspresent on a surface, said method comprising the following steps:

-   -   a) providing        -   at least one detergent component containing at least one            sequestering agent and at least one dispersing agent and at            least one wetting agent, and        -   at least one enzyme component comprising            β-1,6-N-acetylglucosaminidase;    -   b) dissolving or diluting said at least one detergent component        in a solvent;    -   c) dissolving said at least one enzyme component in the solution        formed in step b) to form a composition according to the        invention;    -   or    -   b′) dissolving or diluting said at least one enzyme component in        a solvent,    -   c′) dissolving said at least one detergent component in the        solution formed in step    -   b′), to form a composition according to the invention,    -   d) applying said composition formed in step c) or c′) to said        surface over a predetermined period of time.

Preferably, according to the method according to the invention, saidstep of applying said composition is combined with a step of mechanicalabrasion of said surface with said composition, for example, bymechanical or manual brushing or by application under medium or highpressure. An additional step of mechanical abrasion allows the solutioncomprising said composition in aqueous phase to act on the differentlayers of the biofilms but also to mechanically participate in thebreaking down of the polymer matrix and thus to remove the films of thesurface of the biofilms so the enzyme and other components of saidcomposition better reach the different layers of the biofilms, whichensures optimal treatment of the surfaces in order to effectively removethe biofilms.

Advantageously, the method according to the invention comprises a laststep of applying a biocide to said surface. An additional disinfectingbiocide treatment, following the action of the enzyme solution duringthe step of treating the surface by soaking, allows the destruction ofbacteria liberated at the end of the treatment of said surface to beensured.

Preferably, according to the method according to the invention, saidstep of applying the solution to said composition is a step taking placeover a predetermined period of time ranging between 1 minute and 1 hour,preferably ranging between 3 minutes and 30 minutes, of a solutioncomprising said composition and a previously formed aqueous dilutionphase.

Preferably, the method according to the invention allows a removal of atleast 75%, preferably at least 90%, of the microorganisms present on asurface and protected by a biofilm to be ensured.

This microorganism removal rate, of at least 75%, preferably at least90%, is calculated based on the microorganisms present in a givenenvironment (for example, on the surface of a medical tool or on a floorsurface) before and after a step of treating the surface according tothe invention.

For example, this microorganism removal rate may be calculated by meansof ATP-metric luminometric analysis which allows, by bioluminescence,the number of microorganisms present on a surface to be determined,based on the adenosine triphosphate molecules which were previouslyremoved therefrom, for example, by swabbing. This ATP-metric analysisgiving results expressed in relative light units (RLU), themicroorganism removal rate is defined according to the followingformula: 100−[(RLU value measured after treatment/RLU value measuredbefore treatment)×100].

This microorganism removal rate may also be established by abacteriological analysis consisting of counting the bacterial colonieson the same surface before and after surface treatment carried outaccording to the invention. In the case of bacteriological analyses, themicroorganism removal rate is calculated according to the followingformula: 100−[(CFU value measured after treatment/CFU value measuredbefore treatment)×100].

It is understood that any other technique known to the person skilled inthe art and allowing the number of microorganisms before and aftertreatment of a surface to be determined may also be used in the scope ofthe present invention.

Other embodiments of the method according to the invention are indicatedin the appended claims.

The present invention also includes a kit for removing biofilms presenton a surface, said kit comprising:

-   -   a sample of at least one detergent component in solution or in        solid form containing at least one sequestering agent and at        least one dispersing agent and at least one wetting agent; and    -   a sample of at least one enzyme component in solution or in        solid form comprising β-1,6-N-acetylglucosaminidase.

Advantageously, the kit according to the invention further comprises asample of a biocide in solution or in solid form.

Other embodiments of a kit according to the invention are indicated inthe appended claims.

The present invention also relates to a use of a composition accordingto the invention for removal of biofilms present on a surface bycleaning-in-place, cleaning-by-soaking or by vaporisation of saidsurface.

The present invention also relates to a use of a kit according to theinvention for the removal of biofilms present on a surface bycleaning-in-place, cleaning-by-soaking or by vaporisation of saidsurface.

Other embodiments of a composition or a kit according to the inventionare indicated in the appended claims.

Other features, details and advantages of the invention will becomeclear from the description given below, in a non-limiting manner andreferring to the appended drawings.

FIG. 1 shows the results obtained in terms of removal of the biofilm(reduction of the biofilm %) formed by Staphylococcus epidermis(EP=composition according to document EP2243821; disp.=dispersing agent;seq.=sequestering agent; wet.=wetting agent;β-1,6-N-=β-1,6-N-acetylglucosaminidase).

FIG. 2 shows the results obtained in terms of removal of the biofilm(reduction of the biofilm %) formed by Escherichia coli (EP=compositionaccording to document EP2243821; disp.=dispersing agent;seq.=sequestering agent; wet.=wetting agent;β-1,6-N-=β-1,6-N-acetylglucosaminidase).

FIG. 3 shows the results obtained in terms of removal of the biofilm(reduction of the biofilm %) formed by Pseudomonas fluorescens(EP=composition according to document EP2243821; disp.=dispersing agent;seq.=sequestering agent; wet.=wetting agent;β-1,6-N-=β-1,6-N-acetylglucosaminidase).

FIG. 4 shows the results of the measurements of theβ-1,6-N-acetylglucosaminidase enzyme activities over time based onwhether this enzyme is formulated in the presence of three detergentagents (wetting agent+dispersing agent+sequestering agent) or in thepresence of two of these three detergent agents or based on whether thisenzyme is not formulated in a detergent component.

FIG. 5 shows the results of the measurements of the three cellulasesover time based on whether or not these enzymes are formulated in thepresence of three detergent agents (wetting agent+dispersingagent+sequestering agent) or based on whether this enzyme is notformulated in a detergent component.

EXAMPLES Example 1—Comparative Tests: Effectiveness of a CompositionAccording to the Invention and Effectiveness of a Composition Accordingto Document EP2248321

Comparative tests have been carried out in order to verify that acomposition according to the invention is at least as effective in termsof removing a biofilm as a composition such as that disclosed indocument EP2243821 (cocktail of 3 enzymes in a detergent componentcomprising 3 detergent agents).

The composition according to the invention is as follows:

-   -   Enzyme component: β-1,6-N-acetylglucosaminidase in an amount of        0.005 v/v % with respect to the total volume of the composition;    -   Detergent component: Biorem®A1 in an amount of 0.25 v/v % with        respect to the total volume of the composition.

The composition according to document EP2243821 is as follows:

-   -   Enzyme component: Biorem® 0 in an amount of 0.1 v/v % with        respect to the total volume of the composition;    -   Detergent component: Biorem®A1 in an amount of 0.25 v/v % with        respect to the total volume of the composition.

These components are prepared at room temperature by dilution thereof inwater before carrying out the comparative tests on a biofilm developedfrom Pseudomonas fluorescens.

The first steps of the procedure are similar to the steps of theprotocol of Annexe F of the ISO 15883 standard. In fact, the protocol ofculturing and cleaning the biofilm corresponds to those described inthis standard. The number of biofilms present have, in turn, beenevaluated using the detection kit according to document EP2243821.

Practically, a biofilm has been developed by Pseudomonas fluorescens ina PTFE (Polytetrafluoroethylene) tube with an internal diameter of 6 mm.The tube was filled with a liquid preculture in the exponential growthphase. The preculture was prepared 24 h before the test: a TSB culturemedium was inoculated with the Pseudomonas fluorescens bacterial strainat 30° C. for 12 h. This preculture was then diluted in the TSB culturemedium at 10% in order to obtain an optical density ranging between 300nm and 600 nm. The inoculated culture medium was then circulated in thePTFE tube for 3 days. At the end of this period, the PTFE tube was cutinto sections of a size ranging between 5 and 10 cm on which thedifferent compositions were tested. These different compositions werecirculated for 15 min at a flow of 100 ml/min at a temperature of 45° C.Finally, the number of biofilms present was evaluated by staining eachtube portion with the detection kit disclosed in document EP2537601.

The detection kit is formed of two products, a staining reagent and acleaning reagent. The blue dye present in the staining reagentspecifically adheres to the EPS (extracellular polymeric substances)forming the protective matrix of the biofilm. The surface to be analysedwas sprayed with the staining solution. After 5 min of incubation of thesurface, the excess solution was absorbed. Then, the surface was rinsedusing the cleaning solution of the detection kit which requires anapplication time of 5 min. The surface was finally rinsed with water andanalysed. The presence or absence of biofilm was demonstrated by a bluestain which was darker or lighter according to the number of biofilmsexisting on the surface. An intense blue stain indicates the significantpresence of biofilms.

The results of these tests are presented in Table 1 below.

TABLE 1 Cleaning Staining Before cleaning Intense blue staining WaterIntense blue staining Composition according to document Absence of bluestaining EP2243821 Composition according to the invention Absence ofblue staining

As can be seen from these comparative tests, for the tube portionsinitially strongly contaminated by the biofilm (intense blue stainingbefore cleaning), cleaning with water had no effect on the biofilm andthe tube portions initially strongly contaminated by the biofilmconsequently retained their intense blue staining.

In contrast, whether in the case of cleaning with the compositionaccording to document EP2243821 or in the case of cleaning with thecomposition according to the invention, a total removal of the biofilmwas ensured (absence of blue staining) after cleaning with these twocompositions. This thus indicates that a composition according to theinvention allows the biofilms to be removed in a manner at leastequivalent to the composition according to document EP2243821 but byonly using a single enzyme, i.e. β-1,6-N-acetylglucosaminidase, in placeof three enzymes.

Example 2—Tests of Effectiveness (in Terms of Removal of the Biofilm) ofa Composition According to the Invention Depending on Whether theDetergent Component Simultaneously Comprises a Wetting Agent, aDispersing Agent and a Sequestering Agent or a Combination of Two ofthese Three Detergent Agents

Effectiveness tests were carried out on the biofilm developed bydifferent bacterial strains, namely Staphylococcus epidermis,Escherichia coli and Pseudomonas fluorescens strains.

The biofilms were developed in laboratories under static conditions inmulti-well plates and the effectiveness of β-1,6-N-acetylglucosaminidase(12.5 ppm of the active enzyme in the composition, which corresponds to0.00125 v/v % with respect to the total volume of the composition) wasevaluated on these biofilms following their formulation in the followingdetergent components:

-   -   wetting agent+sequestering detergent+dispersing agent=Biorem®A1        in an amount of 1 v/v % with respect to the total volume of the        composition;    -   wetting agent of Biorem®A1 in an amount of 0.22 v/v % with        respect to the total volume of the composition+sequestering        agent of Biorem®A1 in an amount of 0.25 v/v % with respect to        the total volume of the composition;    -   wetting agent of Biorem®A1 in an amount of 0.22 v/v % with        respect to the total volume of the composition+dispersing agent        of Biorem®A1 in an amount of 0.085 v/v % with respect to the        total volume of the composition;    -   sequestering agent of Biorem®A1 in an amount of 0.25 v/v % with        respect to the total volume of the composition+dispersing agent        of Biorem®A1 in an amount of 0.85 v/v % with respect to the        total volume of the composition;

At the same time, β-1,6-N-acetylglucosaminidase (12.5 ppm of the activeenzyme in the composition which corresponds to 0.00125 v/v % withrespect to the total volume of the composition) was tested alone (notformulated in a detergent component but in aqueous solution) and theeffectiveness of a composition according to document EP2243821(Biorem®A1) at 1 v/v % with respect to the total volume of thecomposition and Biorem 10® at 0.025 v/v % with respect to the totalvolume of the composition).

A. Preparation of the biofilms

Bacterial colonies were obtained by separate sowing of each of thestrains (Staphylococcus epidermis, Escherichia coli or Pseudomonasfluorescens) in a non-specialised solid medium (PCA) then incubated for18 h at 37° C. Then, some bacterial colonies (around 20 colonies) weresuspended in their respective appropriate culture medium. Moreparticularly, the bacterial colonies were suspended in the followingculture media:

-   -   Staphylococcus epidermis: suspension of approximately 20        colonies in 2 ml of TGN (30 g/1 of Tryptic Soy Broth+1 g/l        glucose+2 gl/NaCl);    -   Escherichia coli: suspension of approximately 20 colonies in 2        ml of LGB (15 g/l of peptone water+5 g/l yeast extract+10 g/l        glucose);    -   Pseudomonas fluorescens: suspension of approximately 20 colonies        in 2 ml of TSB (30 g/1 of Tryptic Soy Broth);

Each of these suspensions were adjusted by addition of culture medium(TGN or LBG or TSB) in order to measure an optical density (O.D.) of0.05 to 620 nm, then the bacterial suspensions were placed in the wellsof multi-well plates in an amount of 100 μl per well for Staphylococcusepidermis, 220 μl per well for Escherichia coli and 220 μl per well forPseudomonas fluorescens before incubation for 24 h at 37° C. forStaphylococcus epidermis, for 48 h at 30° C. for Escherichia coli andfor 48 h at 30° C. for Pseudomonas fluorescens. Naturally, for each ofthe bacterial strains tested, some wells were only filled with theculture medium in order to form negative controls.

B. Enzyme Treatment of the Biofilms Formed and Quantification of theRemoval of the Biofilms

Before treating the biofilms formed with various compositions includinga composition according to the invention, the wells of the multi-wellplates were emptied of culture media having allowed the establishmentand the development of the different biofilms. The treatment of thebiofilms itself was carried out by filling the wells with 1 ml of thecleaning solutions (compositions), with softened water being added onlyto the two positive control wells, the two negative control wellsstaying empty, in turn. Following these fillings, incubation at 45° C.for 15 min was observed (duration of cleaning to remove the biofilms).

After this incubation period, the wells were emptied of the cleaningsolutions and rinsed using a sterile saline solution at 0.9% beforedrying the plates for 1 h at 60° C. Following this drying, 0.5 ml ofcrystal violet at 0.1% (powder diluted in demineralised water) was addedto each well before incubation for 15 min at room temperature. Followingthe 15 min of incubation at room temperature, the wells were emptied ofthe crystal violet solution, rinsed twice with distilled water, thendried with absorbent paper if necessary. 1 ml of acetic acid at 33% wasthen added to each well before incubation for 1 h at room temperaturebefore undertaking a reading of the plates at a wavelength of 570 nm(measurement of absorbance by spectrophotometry—Thermo Spectronic UV1).The reduction of the biofilm quantity was calculated with respect to anuncleaned control and a control not containing biofilm—the crystalviolet (CV) is a cationic dye which will be bound in a non-specific wayto the negatively charged constituents of the biofilm and stain themblue. The results of the absorbance measurement of the crystal violetthen allow the biofilm still present after cleaning to be quantified.

The results obtained are presented in FIGS. 1, 2 and 3, respectively forthe biofilms formed by Staphylococcus epidermis, Escherichia coli orPseudomonas fluorescens.

As can be seen, whatever the biofilm considered (formed by S. epidermisor by E. coli or by P. fluorescens), a composition according to theinvention (β-1,6-N-+disp.+seq.+wet.) allows the biofilms to be removedin a significantly superior way with respect to the other compositionstested. This shows that the three detergent agents have an unexpectedsynergic effect allowing the action of β-1,6-N-acetylglucosaminidase interms of removal of the biofilms to be improved. In fact, whenβ-1,6-N-acetylglucosaminidase is formulated in a detergent componentonly comprising two of the three detergent agents, it is observed thatthe removal of the biofilm lessens. The same observation can be madewith a composition according to document EP2243821 which does not allowthe biofilms to be removed as effectively in comparison with acomposition according to the invention.

Example 3—Enzyme Activity of β-1,6-N-Acetylglucosaminidase Depending onWhether the Detergent Component Simultaneously Comprises a WettingAgent, a Dispersing Agent and a Sequestering Agent or a Combination ofTwo of these Three Detergent Agents

The enzyme activity over time of β-1,6-N-acetylglucosaminidase wasmeasured using the 4-nitrophenyl-N-acetyl-b-D-glucosaminide substrate.This substrate releases a yellow compound when it is hydrolysed byβ-1,6-N-acetylglucosaminidase. The higher the hydrolysis rate, thehigher the concentration of yellow compound and the more active theβ-1,6-N-acetylglucosaminidase. The hydrolysis rate is quantified bymeasuring the absorbance of the solution at 405 nm and allows the enzymeactivity of β-1,6-N-acetylglucosaminidase to be judged.

Generally, the enzyme activity over time ofβ-1,6-N-acetylglucosaminidase was measured according to the followingsteps:

-   -   a) β-1,6-N-acetylglucosaminidase was mixed in the different        compositions to be tested (see below) and the thus-obtained        solutions were maintained at a temperature of 40° C. for 2 h in        order to simulate the cleaning conditions;    -   b) samples of these solutions were removed at t=0 (t₀) and t=2 h        (t_(2 h)) in order to measure the enzyme activity of        β-1,6-N-acetylglucosaminidase over time.

More particularly, the following procedure was followed:

-   -   a) a buffer solution at 5.9 pH was prepared: 50 mM sodium        phosphate+5 mM 4-nitrophenyl-N-acetyl-b-D-glucosaminide+100 mM        NaCl;    -   b) the solution obtained in a) was heated to 37° C. and        distributed in an amount of 1 ml in Eppendorfs®;    -   c) 100 μl of the compositions whose enzyme activity over time        must be measured (including a composition according to the        invention) was placed in the Eppendorfs® of point b);    -   d) the concentration of β-1,6-N-acetylglucosaminidase was        adjusted by dilution with the phosphate buffer if necessary in        order to obtain a concentration of approximately 3.7 μg/ml of        β-1,6-N-acetylglucosaminidase;    -   e) the reaction was stopped by adding 5 μl of 10N NaOH after 5        min;    -   f) the absorbance was measured at 405 nm (-Thermo Spectronic        UV1) after 5 minutes (at t₀+5 min and t_(2 h)+5 min), a negative        control (blank) being prepared with water only.

More particularly, the enzyme activity of β-1,6-N-acetylglucosaminidasewas measured over time in the following compositions:

-   -   wetting agent+sequestering agent+dispersing agent (=Biorem®A1 at        0.25 v/v % with respect to the total volume of the        composition)+β-1,6-N-acetylglucosaminidase at 7.4 μg/ml;    -   wetting agent of Biorem®A1 in an amount of 0.055 v/v % with        respect to the total volume of the composition+sequestering        agent of Biorem®A1 in an amount of 0.0625 v/v % with respect to        the total volume of the        composition+β-1,6-N-acetylglucosaminidase at 7.4 μg/ml;    -   wetting agent of Biorem®A1 in an amount of 0.055 v/v % with        respect to the total volume of the composition+dispersing agent        of Biorem®A1 in an amount of 0.021 v/v % with respect to the        total volume of the composition+β-1,6-N-acetylglucosaminidase at        7.4 μg/ml;    -   sequestering agent of Biorem®A1 in an amount of 0.0625 v/v %        with respect to the total volume of the composition+dispersing        agent of Biorem®A1 in an amount of 0.021 v/v % with respect to        the total volume of the        composition+β-1,6-N-acetylglucosaminidase at 7.4 μg/ml;    -   β-1,6-N-acetylglucosaminidase at 7.4 μg/ml in aqueous solution.

The results obtained are shown in FIG. 4 and expressed in % of theβ-1,6-N-acetylglucosaminidase activity measured for a composition notcomprising detergent component (relative enzyme activity). As can beseen, from the start at t₀, that is from the moment whenβ-1,6-N-acetylglucosaminidase is formulated in a detergent componentsimultaneously comprising a wetting agent, a sequestering agent and adispersing agent, its enzyme activity is strongly and significantlypotentiated/increased with respect to the enzyme activities measured forβ-1,6-N-acetylglucosaminidase when it is formulated in a detergentcomponent only comprising two of the three detergent agents.Subsequently, over time and in particular after 2 h, a significantlysuperior enzyme activity of β-1,6-N-acetylglucosaminidase is observedfor a composition according to the invention with respect to the enzymeactivities measured for β-1,6-N-acetylglucosaminidase when it isformulated in a detergent component only containing two of the threedetergent agents.

This shows the synergic effect of the three detergent effects whichtogether, in a significant manner, potentiate/increase the enzymeactivity of β-1,6-N-acetylglucosaminidase which is, consequently, evenmore effective over time.

As a comparison with β-1,6-N-acetylglucosaminidase, three cellulases(other enzymes of another family—Celluclast® 1.5l, Carezyme® 4500l,Viscozyme® 120l) were also tested in terms of enzyme activity over timeafter formulation in Biorem®A1 as detergent component in an amount of0.25 v/v % with respect to the total volume of the composition.

In particular, the enzyme activity of the three cellulases was measuredaccording to the endo-cellulase procedure (CELLG5® method) of theMegazyme® kit by observing the procedure recommended by themanufacturer. The enzyme activities of the cellulases alone or thecellulases formulated in a detergent component according to theinvention were then compared.

The amount of cellulase in the composition was set up in order to be ina concentration range which is in the measurement range of thecommercial kit. Preliminary tests were carried out for each of thecellulases tested and an appropriate concentration for measurement wasselected for each cellulase.

FIG. 5 shows the results obtained and it can be seen that, in contrastto the results obtained for β-1,6-N-acetylglucosaminidase, the samedetergent component (wetting agent+dispersing agent+sequestering agent)absolutely does not have the same effect on the cellulases. In fact, nopotentiation/increase of enzyme activity was directly observed followingthe formulation of each of the cellulases in the detergent component.Furthermore, for these three cellulases, no potentiation/increase ofenzyme activity was maintained over time for this detergent component.Quite the contrary, for Cellulase 2, its formulation in a detergentcomponent identical to that of a composition according to the inventionsignificantly decreased the enzyme activity. This further enforced thesurprising effect observed with a composition according to the inventionwhere β-1,6-N-acetylglucosaminidase, in contrast to these threecellulases, showed a significantly increased enzyme activity when it wasin the presence of three detergent agents (wetting agent+dispersingagent+sequestering agent).

It is understood that the present invention is in no way limited to theembodiments described above and that modifications may be appliedwithout departing from the scope of the appended claims.

1. Composition for the removal of biofilms, said composition comprisingat least one enzyme component and at least one detergent component,characterised in that the at least one enzyme component comprisesβ-1,6-N-acetylglucosaminidase and in that said detergent componentcontains at least one wetting agent, at least one sequestering agent andat least one dispersing agent.
 2. Composition for the removal ofbiofilms according to claim 1, characterised in that said at least oneenzyme component comprises at least one additional enzyme chosen fromthe group constituted of group constituted of α-polysaccharidases(lactase, amylase, α-glucosidase), β-polysaccharidases (cellulase,hemi-cellulase, β-glucanase, arabanase, pectinase, chitinase, xylanase,dextranase, lysozyme, pullulanase, β-glucisidase, mannanase),oxidoreductases (laccase), lyases (lyase pectate), transferases,proteases and peptidases (metalloprotease, serine-proteases,exopeptidase, endoprotease, cysteine protease), lipases and esterases(lysophospholipase, phospholipase).
 3. Composition for the removal ofbiofilms according to claim 1, characterised in that it has a pH rangingbetween 5 and
 11. 4. Composition for the removal of biofilms accordingto claim 1, characterised in that said at least one detergent componentcomprises a proportion of sequestering agent between 1 and 10 wt %, aproportion of dispersing agent between 1 and 10 wt % and a proportion ofwetting agent between 1 and 30 wt % with respect to the total weight ofthe detergent component.
 5. Composition for the removal of biofilmsaccording to claim 1, characterised in that said at least one enzymecomponent and said at least one detergent component are in solution in asolvent, for example, in solution in a solvent in order to form avaporisable solution.
 6. Composition for the removal of biofilmsaccording to claim 1, characterised in that said at least one enzymecomponent and said at least one detergent component are in a solid form,for example, in the form of a lyophilisate, a powder, granules, or inany other form soluble in a solvent.
 7. Method for removing biofilmspresent on a surface, said method comprising the following steps: a)providing at least one detergent component containing at least onesequestering agent and at least one dispersing agent and at least onewetting agent, and at least one enzyme component comprisingβ-1,6-N-acetylglucosaminidase; b) dissolving or diluting said at leastone detergent component in a solvent; c) dissolving said at least oneenzyme component in the solution formed in step b) to form a compositionaccording to claim 1; or b′) dissolving or diluting said at least oneenzyme component in a solvent, c′) dissolving said at least onedetergent component in the solution formed in step b′), to form acomposition according to claim 1, d) applying said composition formed instep c) or c′) to said surface over a predetermined period of time. 8.Method according to claim 7, characterised in that said step of applyingsaid composition is combined with a step of mechanical abrasion of saidsurface with said composition by mechanical or manual brushing or byapplication under medium or high pressure.
 9. Method according to claim7, characterised in that it comprises a last step of applying a biocideto said surface.
 10. Kit for the removal of biofilms present on asurface, said kit comprising: a sample of at least one detergentcomponent in solution or in a solid form containing at least onesequestering agent and at least one dispersing agent and at least onewetting agent; and a sample of at least one enzyme component in solutionor in a solid form comprising β-1,6-N-acetylglucosaminidase.
 11. Kitaccording to claim 10, characterised in that it further comprises asample in solution or in a solid form of a biocide.
 12. Use of acomposition according to claim 1 for removal of biofilms present on asurface by cleaning-in-place, cleaning-by-soaking orcleaning-by-vaporisation of said surface.
 13. Use of a kit according toclaim 10 for removal of biofilms present on a surface bycleaning-in-place, cleaning-by-soaking or cleaning-by-vaporisation ofsaid surface.